| Viruses must typically penetrate protective mucus secretions to establish reliable infection in the respiratory, gastrointestinal, and female reproductive tract. Mucus gels may trap viruses that are either too large to diffuse through the openings in their mesh, or that adhere to their mesh elements. Although the mucus layer is often overlooked, understanding virus transport in mucus is critical to: (i) prevention and/or minimization of disease transmission in mucosal tissues, and (ii) the design of improved vectors for gene therapy that can efficiently penetrate mucus barriers.;Using high resolution multiple particle tracking, we demonstrate that human mucus often provides a formidable barrier to pathogenic viruses. Cervicovaginal mucus from donors with healthy lactobacilli dominated flora (CVM; pH∼4) is highly efficient at trapping Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) and Human Papilloma Virus (HPV). However, at any given time, 1 in 3 women in the United States have bacteriovaginosis (BV), a condition which leads to waterier, less viscoelastic and more neutral (pH 5-6) vaginal secretions. When CVM was partially neutralized to mimic the neutral secretions of BV, both HIV & HSV rapidly penetrated the mucus barrier. BV has been associated with increased susceptibility to numerous sexually transmitted diseases, including HSV and HIV. Therefore, methods that maintain the natural acidity of CVM may be useful in vaginal microbicide development.;To be useful for gene therapy of Cystic Fibrosis (CF), viral vectors must penetrate lung airway mucus (sputum) prior to reaching airway epithelial cells. We show that purulent sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus and adeno-associated virus serotype 5. Methods that improve vector penetration across the sputum layer are likely needed if aerosol gene therapy for CF is to be successful. Toward this end, we investigated whether N-acetylcysteine (NAC) or recombinant human DNase (rhDNase), two clinically used mucolytics, could aid vector penetration rates in CF sputum. We found that treatment of sputum with NAC improved both adenovirus and AAV transport by an average of 7.6-fold and 19-fold, respectively. However, transport of neither gene therapy vector was significantly improved in rhDNAse treated sputum samples.;The ability to manipulate and engineer the viral surface for enhanced efficiency is likely necessary to overcome many barriers, including the presence of pre-existing immunity and transduction of non-target cells. Although single amino acid changes and peptide insertions at select sites have been explored for AAV previously, we applied a novel directed evolution approach to addresses the tolerance of AAV to small deletions and tandem duplications. A large, diverse library of >105 members containing deletions and tandem duplications throughout the viral capsid of AAV5 was created and four unique mutants were identified that maintain the ability to form viral particles. This approach may be useful for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands. |
